Nucleic acids encoding B7-H4, a T cell immunoregulatory molecule

ABSTRACT

The invention provides novel B7-H3 and B7-H4 polypeptides useful for co-stimulating T cells, isolated nucleic acid molecules encoding them, vectors containing the nucleic acid molecules, and cells containing the vectors. Also included are methods of making and using these co-stimulatory polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/120,927, filed May 2, 2005, now U.S. Pat. No. 7,622,565 which iscontinuation of U.S. application Ser. No. 09/915,789, filed Jul. 26,2001, now U.S. Pat. No. 6,891,030, issued May 10, 2005, which claimspriority to U.S. Provisional Application No. 60/220,991, filed Jul. 27,2000. The disclosures of U.S. application Ser. No. 11/120,927, U.S.application Ser. No. 09/915,789, and U.S. Provisional Application No.60/220,991 are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

The invention is generally in the field of immunoregulation, andspecifically T cell response regulation.

Mammalian T lymphocytes recognize antigenic peptides bound to majorhistocompatibility complex (MHC) molecules on the surface of antigenpresenting cells (APC). The antigenic peptides are generated byproteolytic degradation of protein antigens within the APC. Theinteraction of the T cells with the APC and the subsequent response ofthe T cells are qualitatively and quantitatively regulated byinteractions between cell surface receptors on the T cells with bothsoluble mediators and ligands on the surface of APC.

SUMMARY OF THE INVENTION

The invention is based on the cloning of three human cDNA moleculesencoding three novel polypeptides that co-stimulate T cell responses andon the functional characterization of the polypeptides that the cDNAmolecules encode. Two of these novel co-stimulatory polypeptides, theB7-H3 polypeptides, differ from each other by a single amino acidresidue (position number 166). The B7-H3 polypeptide with serine atposition 166 is designated herein B7-H3.1 (SEQ ID NO:1) and that withproline at position 166 is designated B7-H3.2 (SEQ ID NO:3). Text thatrefers to B7-H3 without specifying B7-H3.1 or B7-H3.2 is pertinent toboth polypeptides. The third novel co-stimulatory polypeptide, B7-H4, isencoded by a separate gene. The invention features DNA moleculesencoding the B7-H3 and B7-H4 polypeptides, functional fragments of thepolypeptides, and fusion proteins containing the polypeptides orfunctional fragments of the polypeptides, B7-H3 and B7-H4 and functionalfragments of both, vectors containing the DNA molecules, and cellscontaining the vectors. Also included in the invention are antibodiesthat bind to the B7-H3 and B7-H4 polypeptides. The invention features invitro, in vivo, and ex vivo methods of co-stimulating T cell responses,methods of screening for compounds that inhibit or enhance T cellresponses, and methods for producing the polypeptides and fusionproteins.

Specifically the invention features an isolated DNA including: (a) anucleic acid sequence that (i) encodes a polypeptide with the ability toco-stimulate a T cell, and (ii) hybridizes under stringent conditions tothe complement of a sequence that encodes a polypeptide with an aminoacid sequence with SEQ ID NO:1, or SEQ ID NO:3, or SEQ ID NO:5; or (b) acomplement of this nucleic acid sequence. The polypeptide-encodingnucleic acid sequence included in the isolated DNA will be at least 10bp, 15 bp, 25 bp, 50 bp, 75 bp, 100 bp, 125 bp, 150 bp, 175 bp, 200 bp,250 bp, 300 bp, 350 bp, 400 bp, 450 bp, 500 bp, 550 bp, 600 bp, 650 bp,700 bp, 750 bp, 800 bp, 840 bp, 850 bp, 900 bp, or 940 bp long. Thenucleic acid sequence can encode a B7-H3 polypeptide that includes anamino acid sequence (a) extending from amino acid 27, 28, 29, 30, 31,32, 33, or 34 to amino acid 316 of either SEQ ID NO:1 or SEQ ID NO:3, or(b) of SEQ ID NO:1 or SEQ ID NO:3. Alternatively, the nucleic acidsequence can have a nucleotide sequence with SEQ ID NO:2 or SEQ ID NO:4.The nucleic acid sequence can encode a B7-H4 polypeptide that includesan amino acid sequence with SEQ ID NO:5 or it can have a nucleotidesequence with SEQ ID NO:6. The nucleic acid sequence can also encodefunctional fragments of these B7-H3 or B7-H4 polypeptides.

The invention also embodies an isolated B7-H3 polypeptide encoded by aDNA that includes a nucleic acid sequence that (i) encodes a polypeptidewith the ability to co-stimulate a T cell and (ii) hybridizes understringent conditions to the complement of a sequence that encodes apolypeptide with an amino acid sequence with SEQ ID NO:1 or SEQ ID NO:3.The B7-H3 polypeptide can include an amino acid sequence starting atresidue 27, 28, 29, 30, 31, 32, 33 or 34 and extending to 361 of SEQ IDNO:1 or SEQ ID NO:3. The invention also encompasses B7-H3 polypeptidesthat include an amino acid sequence with SEQ ID NO:1 or SEQ ID NO:3, oreither of these amino acid sequences but differing solely by one or more(e.g., two, three, four, five, six, seven, eight, nine, ten, 12, 15, 20,25, 30, 35, 40, 50, 60, 80, or 100) conservative substitutions.

The invention also includes an isolated B7-H4 polypeptide encoded by DNAthat includes a nucleic acid sequence that (i) encodes a polypeptidewith the ability to co-stimulate a T cell and (ii) hybridizes understringent conditions to the complement of a sequence that encodes apolypeptide with an acid sequence with SEQ ID NOS:5. The B7-H4polypeptide can include an amino acid sequence starting at residue 27,28, 29, 30, 31, 32, 33, or 34 and extending to residue 282 of SEQ ID NO:5 or this amino acid sequence but differing solely by one or more (e.g.,two, three, four, five, six, seven, eight, nine, ten, 12, 15, 20, 25,30, 35, 40, 50, 60, 80 or 100) conservative substitutions.

Also encompassed by the invention are functional fragments of any of theabove polypeptides.

The polypeptides of the invention include fusion proteins containing afirst domain and at least one additional domain. The first domain can beany of the B7-H3 or B7-H4 polypeptides described above or a functionalfragment of any of these polypeptides. The at least one additionaldomain can be a heterologous targeting or leader sequence, an amino acidsequence that facilitates purification, detection, or solubility of thefusion protein. The second domain can be, for example, all or part of animmunoglobulin (Ig) heavy chain constant region. Also included areisolated nucleic acid molecules encoding the fusion proteins.

The invention features vectors containing any of the DNAs of theinvention and nucleic acid molecules encoding the fusion proteins of theinvention. The vectors can be expression vectors in which the nucleicacid coding sequence or molecule is operably linked to a regulatoryelement which allows expression of the nucleic acid sequence or moleculein a cell. Also included in the invention are cells (e.g., mammalian,insect, yeast, fungal, or bacterial cells) containing any of the vectorsof the invention.

Another embodiment of the invention is a method of co-stimulating a Tcell that involves contacting the T cell with any of the B7-H3 or B7-H4polypeptides of the invention, functional fragments thereof, or fusionproteins of the invention; these 3 classes of molecule are, forconvenience, designated “B7-H3 agents” or “B7-H4 agents.” The contactingcan be by, for example, culturing any of these B7-H3 or B7-H4 agentswith the T cell in vitro. Alternatively, the T cell can be in a mammaland the contacting can be by, for example, administering any of theB7-H3 or B7-H4 agents to the mammal or administering a nucleic acidencoding the B7-H3 or B7-H4 agent to the mammal. In addition, the methodcan be an ex vivo procedure that involves: providing a recombinant cellwhich is the progeny of a cell obtained from the mammal and has beentransfected or transformed ex vivo with a nucleic acid encoding any ofthe B7-H3 or B7-H4 agents so that the cell expresses the B7-H3 or B7-H4agent; and administering the cell to the mammal. In this ex vivoprocedure, the cell can be an antigen presenting cell (APC) thatexpresses the B7-H3 agent or B7-H4 agent on its surface. Furthermore,prior to administering to the mammal, the APC can be pulsed with anantigen or an antigenic peptide. In addition, the cell obtained from themammal can be a tumor cell. In any of these methods of the invention,the B7-H3 or B7-H4 agents can co-stimulate the production ofinterferon-γ by the T cell.

Also embodied by the invention is a method of co-stimulating a T cell inwhich the T cell is contacted with: (a) a first co-stimulatorypolypeptide that can be either (i) B7-H1, (ii) B7-H2, (iii) B7-H3, (iv)B7-H4, (v) a functional fragment of any of (i)-(iv), or (vi) any of(i)-(v) but with one or more conservative substitutions; and (b) one ormore additional co-stimulatory polypeptides that can be either (vi)B7-1, (vii) B7-2, (viii) B7-H1, (ix) B7-H2, (x) B7-H3, (xi) B7-H4, (xii)a functional fragment of any of (vi)-(xi), or (xii) any of (vi)-(xii)but with one or more conservative substitutions. The contacting can beby, for example, culturing the first co-stimulatory polypeptide and theone or more additional co-stimulatory polypeptides with the T cell invitro. Alternatively, the T cell can be in a mammal and the contactingcan be by, for example, administering the first co-stimulatorypolypeptide and the one or more additional co-stimulatory polypeptidesto the mammal. In addition, contacting of a T cell in a mammal can be byadministering one or more nucleic acids encoding the firstco-stimulatory polypeptide and the one more additional co-stimulatorypolypeptides to the mammal. The method can also be an ex vivo procedurethat, for example, involves: providing a recombinant cell which is theprogeny of a cell obtained from the mammal and which has beentransfected or transformed ex vivo with one or more nucleic acidsencoding the first co-stimulatory polypeptide and the one or moreadditional polypeptides so that the cell expresses the firstco-stimulatory polypeptide and the one or more additional co-stimulatorypolypeptides; and administering the cell to the mammal. Alternatively,the ex vivo procedure can involve: providing a first recombinant cellwhich is the progeny of a cell obtained from the mammal and which hasbeen transfected or transformed ex vivo with a nucleic acid encoding thefirst co-stimulatory polypeptide; providing one or more additionalrecombinant cells each of which is the progeny of a cell obtained fromthe mammal and each of which has been transfected or transformed ex vivowith a nucleic acid encoding one of the additional one or moreco-stimulatory polypeptides; and administering the first cell and theone or more additional cells to the mammal. The recombinant cells usedin the any of the ex vivo procedures can be antigen presenting cells(APC) and they can express the first co-stimulatory polypeptide and/orthe one or more additional co-stimulatory polypeptides on theirsurfaces. Prior to the administering, APC can be pulsed with an antigenor an antigenic peptide. In addition, the cell obtained from the mammalcan be a tumor cell.

In any of the above methods of co-stimulating a T cell, the mammal canbe suspected of having, for example, an immunodeficiency disease, aninflammatory condition, or an autoimmune disease.

The invention includes a method of identifying a compound that inhibitsan immune response. The method involves: providing a test compound;culturing, together, the compound, one or more B7-H3 or B7-H4 agents, aT cell, and a T cell activating stimulus; and determining whether thetest compound inhibits the response of the T cell to the stimulus, as anindication that the test compound inhibits an immune response. Theinvention also embodies a method of identifying a compound that enhancesan immune response. The method involves: providing a test compound;culturing, together, the compound, one or more of B7-H3 or B7-H4 agents,a T cell, and a T cell activating stimulus; and determining whether thetest compound enhances the response of the T cell to the stimulus, as anindication that the test compound enhances an immune response. In boththese methods, the stimulus can be, for example, an antibody that bindsto a T cell receptor or a CD3 polypeptide. Alternatively, the stimuluscan be an alloantigen or an antigenic peptide bound to a majorhistocompatibility complex (MHC) molecule on the surface of an antigenpresenting cell (APC). The APC can be transfected or transformed with anucleic acid encoding the B7-H3 or B7-H4 agent and the B7-H3 or B7-H4agent can be expressed on the surface of the APC.

The invention also features an antibody (e.g., a polyclonal or amonoclonal antibody) that binds specifically to one of the B7-H3 orB7-H4 polypeptides of the invention, e.g., the polypeptide with SEQ IDNO:1, SEQ ID NO:3, or SEQ ID NO:5.

The invention also features a method of producing any of the B7-H3 orB7-H4 polypeptides of the invention, functional fragments thereof, orfusion proteins of the invention. The method involves culturing a cellof the invention and purifying the relevant B7-H3 or B7-H4 protein fromthe culture.

“Polypeptide” and “protein” are used interchangeably and mean anypeptide-linked chain of amino acids, regardless of length orpost-translational modification. The invention also features B7-H3 andB7-H4 polypeptides with conservative substitutions. Conservativesubstitutions typically include substitutions within the followinggroups: glycine and alanine; valine, isoleucine, and leucine; asparticacid and glutamic acid; asparagine, glutamine, serine and threonine;lysine, histidine and arginine; and phenylalanine and tyrosine.

The term “isolated” polypeptide or peptide fragment as used hereinrefers to a polypeptide or a peptide fragment which either has nonaturally-occurring counterpart (e.g., a peptidomimetic), or has beenseparated or purified from components which naturally accompany it,e.g., in tissues such as pancreas, liver, spleen, ovary, testis, muscle,joint tissue, neural tissue, gastrointestinal tissue, or body fluidssuch as blood, serum, or urine. Typically, the polypeptide or peptidefragment is considered “isolated” when it is at least 70%, by dryweight, free from the proteins and naturally-occurring organic moleculeswith which it is naturally associated. Preferably, a preparation of apolypeptide (or peptide fragment thereof) of the invention is at least80%, more preferably at least 90%, and most preferably at least 99%, bydry weight, the polypeptide (or the peptide fragment thereof),respectively, of the invention. Thus, for example, a preparation ofpolypeptide x is at least 80%, more preferably at least 90%, and mostpreferably at least 99%, by dry weight, polypeptide x. Since apolypeptide that is chemically synthesized is, by its nature, separatedfrom the components that naturally accompany it, the syntheticpolypeptide or nucleic acid is “isolated.”

An isolated polypeptide (or peptide fragment) of the invention can beobtained, for example, by extraction from a natural source (e.g., fromhuman tissues or bodily fluids); by expression of a recombinant nucleicacid encoding the peptide; or by chemical synthesis. A peptide that isproduced in a cellular system different from the source from which itnaturally originates is “isolated,” because it will be separated fromcomponents which naturally accompany it. The extent of isolation orpurity can be measured by any appropriate method, e.g., columnchromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

An “isolated DNA” means DNA free of the genes that flank the gene ofinterest in the genome of the organism in which the gene of interestnaturally occurs. The term therefore includes a recombinant DNAincorporated into a vector, into an autonomously replicating plasmid orvirus, or into the genomic DNA of a prokaryote or eukaryote. It alsoincludes a separate molecule such as: a cDNA where the correspondinggenomic DNA has introns and therefore a different sequence; a genomicfragment; a fragment produced by polymerase chain reaction (PCR); arestriction fragment; a DNA encoding a non-naturally occurring protein,fusion protein, or fragment of a given protein; or a nucleic acid whichis a degenerate variant of a naturally occurring nucleic acid. Inaddition, it includes a recombinant nucleotide sequence that is part ofa hybrid gene, i.e., a gene encoding a fusion protein. Also included isa recombinant DNA that includes a portion of SEQ ID NO:2, SEQ ID NO:4,or SEQ ID NO:6. From the above it will be clear that an isolated DNAdoes not include a restriction fragment containing all or part of a genethat flanks the gene of interest in the genome of the organism in whichthe gene of interest naturally occurs. Furthermore, an isolated DNA doesnot mean a DNA present among hundreds to millions of other DNA moleculeswithin, for example, cDNA or genomic DNA libraries or genomic DNArestriction digests in, for example, a restriction digest reactionmixture or an electrophoretic gel slice.

As used herein, a polypeptide that “co-stimulates” a T cell is apolypeptide that, upon interaction with a cell-surface molecule on the Tcell, enhances the response of the T cell. The T cell response thatresults from the interaction will be greater than the response in theabsence of the polypeptide. The response of the T cell in the absence ofthe co-stimulatory polypeptide can be no response or it can be aresponse significantly lower than in the presence of the co-stimulatorypolypeptide. It is understood that the response of the T cell can aneffector, helper, or suppressive response.

As used herein, the term “co-stimulatory” polypeptide or moleculeincludes molecules such as B7-1, B7-2, B7-H1, B7-H2, B7-H3, B7-H4,4-1BB, OX40, and herpes virus entry mediator (HVEM). As used herein, an“activating stimulus” is a molecule that delivers an activating signalto a T cell, preferably through the antigen specific T cell receptor(TCR). The activating stimulus can be sufficient to elicit a detectableresponse in the T cell. Alternatively, the T cell may requireco-stimulation (e.g., by a B7-H3 or B7-H4 polypeptide) in order torespond detectably to the activating stimulus. Examples of activatingstimuli include, without limitation, antibodies that bind to the TCR orto a polypeptide of the CD3 complex that is physically associated withthe TCR on the T cell surface, alloantigens, or an antigenic peptidebound to a MHC molecule.

As used herein, a “fragment” of a B7-H3 or B7-H4 polypeptide is afragment of the polypeptide that is shorter than the full-lengthpolypeptide. Generally, fragments will be five or more amino acids inlength. An antigenic fragment has the ability to be recognized and boundby an antibody.

As used herein, a “functional fragment” of a B7-H3 or B7-H4 polypeptideis a fragment of the polypeptide that is shorter than the full-lengthpolypeptide and has the ability to co-stimulate a T cell. Methods ofestablishing whether a fragment of an B7-H3 or B7-H4 molecule isfunctional are known in the art. For example, fragments of interest canbe made by either recombinant, synthetic, or proteolytic digestivemethods. Such fragments can then be isolated and tested for theirability to co-stimulate T cells by procedures described herein.

As used herein, “operably linked” means incorporated into a geneticconstruct so that expression control sequences effectively controlexpression of a coding sequence of interest.

As used herein, the term “antibody” refers not only to whole antibodymolecules, but also to antigen-binding fragments, e.g., Fab, F(ab′)₂,Fv, and single chain Fv fragments. Also included are chimericantibodies.

As used herein, an antibody that “binds specifically” to an isolatedB7-H4 polypeptide encoded by a DNA that includes a nucleic acid sequencethat (i) encodes a polypeptide with the ability to co-stimulate a T celland (ii) hybridizes under stringent conditions to the complement of asequence that encodes a polypeptide with an amino acid sequence with SEQID NO:5, is an antibody that does not bind substantially to B7-1, B7-2,B7-H1, B7-H2, or B7-H3.

As used herein, an antibody that “binds specifically” to an isolatedB7-H3 polypeptide that includes an amino acid sequence of amino acidresidue 31 to amino acid residue 316 of SEQ ID NO: 3 is an antibody thatdoes not bind substantially to B7-1, B7-2, B7-H1, B7-H2, or B7-H4. Inaddition, an antibody that binds specifically to B7-H3.1 preferably doesnot substantially bind to B7-H3.2 and vice versa.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. In case of conflict, thepresent document, including definitions, will control. Preferred methodsand materials are described below, although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention. All publications, patentapplications, patents and other references mentioned herein areincorporated by reference in their entirety. The materials, methods, andexamples disclosed herein are illustrative only and not intended to belimiting.

Other features and advantages of the invention, e.g., enhancing immuneresponses in mammalian subjects, will be apparent from the followingdescription, from the drawings and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a depiction of the nucleotide sequence of cDNA encodingB7H-3.1 (SEQ ID NO:2). Nucleotide residue 496 is shown in bold.

FIG. 2 is a depiction of the nucleotide sequence of cDNA encodingB7H-3.2 (SEQ ID NO:4). Nucleotide residue 496 is shown in bold.

FIG. 3 is a depiction of the amino acid sequence of B7-H3.1 (SEQ IDNO:1). Amino acid residue 166 is shown in bold.

FIG. 4 is a depiction of the amino acid sequence of B7-H3.2 (SEQ IDNO:3). Amino acid residue 166 is shown in bold.

FIG. 5A is a depiction of the amino acid sequences of human B7-H3.1 (SEQID NO:1), human B7-1 (SEQ ID NO:15), human B7-2 (SEQ ID NO:16), humanB7-H1 (SEQ ID NO:17), and human B7-2 (SEQ ID NO:18) aligned for optimalhomology. Identical amino acid residues are shaded in bold and conservedamino acid residues are boxed. Conserved cysteine residues are marked *.

FIG. 5B is a depiction of the amino acid sequence of B7-H.3.1 showingthe predicted signal peptide

, IgV-like domain

, IgC-like domain

, transmembrane domain

, and intracellular tail

. Potential N-glycosylation sites are shown in bold (“N”).

FIG. 6A is a pair of fluorescence flow cytometry histograms showing thestaining by an anti-B7-H3 antiserum (open area) or control serum (shadedarea) of 293 cells transfected with either the control pcDNA3.1(−)parental vector (“293/mock”; left histogram) or the pcDNA/B7-H3 vectorcontaining the B7H-3.1 coding region (“293/B7-H3”; right histogram).

FIG. 6B is a series of fluorescence flow cytometry histograms showingthe staining by an anti-B7-H3 antiserum (open area) or control serum(shaded area) of various cell types.

FIG. 7A is a series of fluorescence flow cytometry histograms showingthe ability of B7-H3Ig fusion protein to bind to activated T cells butnot to resting T cells. Unactivated nylon wool-purified T cells andnylon wool-purified T cells activated with PHA (5 μg/ml) for 24 h, 48 h,or 72 h were stained with B7-H3Ig (5 μg; open areas) or control Ig (5μg) prior to analysis.

FIG. 7B is a series of fluorescence flow cytometry histograms showingthe ability of anti-B7-H3 antiserum, CTLA4Ig fusion protein, and ICOSIgfusion protein to bind to 293 cells transfected with expression vectorscontaining either a B7-H3.1 (solid line, shaded area), B7-1 (solid line,open area), or B7-H2 (dotted line, open area) coding sequence.

FIG. 8A is a line graph showing the proliferative response of nylonwool-purified T cells activated by anti-CD3 mAb (coated onto tissueculture well bottoms using a concentration of 40 ng/ml) andco-stimulated by either control Ig, B7-1Ig fusion protein, or B7-H3Igfusion protein coated onto the tissue culture well bottoms at theindicated concentrations.

FIG. 8B is a bar graph showing the proliferative responses of purifiedCD4+ and CD8+ T cells activated by anti-CD3 mAb (coated onto tissueculture well bottoms using a concentration of 40 ng/ml) andco-stimulated by either control Ig, B7-1Ig fusion protein, or B7-H3Igfusion protein, each coated onto tissue culture well bottoms using aconcentration of 10 μg/ml.

FIG. 8C is a line graph showing the cytolytic activity on wild-type624mel tumor target cells of cytotoxic T lymphocytes (CTL) (at theindicated effector to target cell ratios (“E:T”)) generated by culturingnylon wool-purified T cells with 624mel tumor cells transfected witheither the control parental pcDNA3.1(−) expression vector(“Mock/624mel”) or the pcDNA/B7-H3 expression vector containing theB7H-3.1 coding region (“B7-H3/624mel”).

FIG. 9 is a depiction of the nucleotide sequence of cDNA encoding B7-H4(SEQ ID NO:6).

FIG. 10 is a depiction of the amino acid sequence of B7-H4 (SEQ IDNO:5).

FIG. 11 is a depiction of the amino acid sequences of the extracellulardomains of human B7-H4 (SEQ ID NO: 19), human B7-H3.2 (SEQ ID NO:20),human B7-1 (SEQ ID NO:21), human B7-2 (SEQ ID NO:22), human B7-H1 (SEQID NO:23), and human B7-2 (SEQ ID NO:24) aligned for optimal homology.Identical amino acid residues are shaded in bold and conserved aminoacid residues are boxed. Conserved cysteine residues are marked *.

FIG. 12 is a line graph showing the proliferative response (“CPM”) ofnylon wool-purified T cells activated by anti-CD3 mAb (coated ontotissue culture well bottoms using the indicated concentrations) andco-stimulated by either control human IgG (“hIgG”), B7-1Ig fusionprotein, B7-H4Ig fusion protein, or B7-H4hIg coated onto tissue culturewell bottoms at a concentration of 5 μg/ml.

FIG. 13 is a line graph showing the proliferative response (“CPM”) ofnylon wool-purified T cells activated by anti-CD3 mAb (coated ontotissue culture well bottoms using the indicated concentrations) andco-stimulated by either control human IgG (“hIgG”), B7-1Ig fusionprotein, B7-H4Ig fusion protein, or B7-H4hIg fusion protein coated ontotissue culture well bottoms at a concentration of 10 μg/ml.

FIG. 14 is a bar graph showing the relative levels of mRNA encoding 23cytokines in nylon wool purified T cells activated by anti-CD3 mAb(coated onto tissue culture well bottoms at a concentration of 500ng/ml) and co-stimulated by either control human IgG (coated onto tissueculture well bottoms at a concentration of 5 μg/ml) (“hIgG”), B7-1Igfusion protein (coated onto tissue culture well bottoms at aconcentration of 5 μg/ml), or B7-H3Ig fusion protein (coated onto tissueculture well bottoms at a concentration of 5 μg/ml).

DETAILED DESCRIPTION

Using a novel PCR-based strategy, the inventor has identified two cDNAsequences (SEQ ID NOS:2 and 4) corresponding to two alleles of a geneencoding a novel B7-related molecule (B7-H3).

Translation of the cDNA sequences indicated that the two polypeptides(B7-H3.1 and B7-H3.2; SEQ ID NOS:1 and 3, respectively) encoded by thetwo allelic cDNA molecules are type I transmembrane proteins of 316amino acids, each containing an immunoglobulin (Ig) V-like domain, IgC-like domain, a transmembrane domain and a cytoplasmic domain of 30amino acids. Northern blot analysis showed strong expression of the geneencoding B7-H3 in heart, liver, placenta, prostate, testis, uterus,pancreas, small intestine, and colon, and weak expression in brain,skeletal muscle, kidney, and lung. Expression was undetectable inperipheral blood mononuclear cells (PBMC) but was detectable in spleen,lymph nodes, bone marrow, fetal liver, and thymus.

Fluorescence flow cytometry using an antiserum produced by immunizationof mice with a carrier-conjugated peptide corresponding to a hydrophilicregion of B7-H3, indicated no expression of B7-H3 on the majority ofhematopoietic cells. However, less than 3% of resting CD14+ cellsexpressed the molecule on their surfaces. Activation with phorbolmyristic acid (PMA) and ionomycin increased expression on T cells,monocytes, and dendritic cells.

Binding experiments indicated that T cells express a counter-receptorfor B7-H3 that is not CTLA4, ICOS, or CD28.

In vitro experiments with isolated human T cells and aB7-H3.1-containing fusion protein indicated that while B7-H3 had nodirect activity on T cells, it enhanced (“co-stimulated”) CD4+ and CD8+T cell proliferative responses induced by antibody specific for humanCD3.

Using a strategy similar to that used to clone B7-H3 cDNA, a cDNAmolecule containing an open reading frame (orf) encoding another B7homologue (B7-H4) was cloned, the nucleotide sequence of the orf (SEQ IDNO:6) was obtained, and the amino acid sequence of the encoded sequence(SEQ ID NO:5) was derived. B7-H4 is a type I transmembrane protein of282 amino acids and has the same domain structure as B7-H3. Northernblot analysis showed expression of B7-H4 in lymphoid organs such asspleen and thymus. Binding experiments showed that there is acounter-receptor for B7-H4 on activated but not resting T cells. B7-H4,like B7-H3, co-stimulated T cell proliferation.

B7-H3 and B7-H4 can be used as augmenters of immune responses both invivo and in vitro.

Nucleic Acid Molecules

The B7-H3 and B7-H4 nucleic acid molecules of the invention can be cDNA,genomic DNA, synthetic DNA, or RNA, and can be double-stranded orsingle-stranded (i.e., either a sense or an antisense strand). Fragmentsof these molecules are also considered within the scope of theinvention, and can be produced by, for example, the polymerase chainreaction (PCR) or generated by treatment with one or more restrictionendonucleases. A ribonucleic acid (RNA) molecule can be produced by invitro transcription. Preferably, the nucleic acid molecules encodepolypeptides that, regardless of length, are soluble under normalphysiological conditions.

The nucleic acid molecules of the invention can contain naturallyoccurring sequences, or sequences that differ from those that occurnaturally, but, due to the degeneracy of the genetic code, encode thesame polypeptide (for example, the polypeptides with SEQ ID NOS:1, 3 or5). In addition, these nucleic acid molecules are not limited to codingsequences, e.g., they can include some or all of the non-codingsequences that lie upstream or downstream from a coding sequence.

The nucleic acid molecules of the invention can be synthesized (forexample, by phosphoramidite-based synthesis) or obtained from abiological cell, such as the cell of a mammal. Thus, the nucleic acidscan be those of a human, non-human primate (e.g., monkey) mouse, rat,guinea pig, cow, sheep, horse, pig, rabbit, dog, or cat.

In addition, the isolated nucleic acid molecules of the inventionencompass segments that are not found as such in the natural state.Thus, the invention encompasses recombinant nucleic acid molecules (forexample, isolated nucleic acid molecules encoding B7-H3 or B7-H4)incorporated into a vector (for example, a plasmid or viral vector) orinto the genome of a heterologous cell or into the genome of ahomologous cell at a position other than the natural chromosomallocation. Recombinant nucleic acid molecules and uses therefor arediscussed further below.

Certain nucleic acid molecules of the invention are antisense moleculesor are transcribed into antisense molecules. These can be used, forexample, to down-regulate translation of B7-H3 or B7-H4 mRNA within acell.

Techniques associated with detection or regulation of genes are wellknown to skilled artisans and such techniques can be used to diagnoseand/or treat disorders associated with aberrant B7-H3 or B7-H4expression. Nucleic acid molecules of the invention are discussedfurther below in the context of their therapeutic utility.

A B7-H3 or B7-H4 family gene or protein can be identified based on itssimilarity to the relevant B7-H3 or B7-H4 gene or protein, respectively.For example, the identification can be based on sequence identity. Theinvention features isolated nucleic acid molecules which are at least50% (or 55%, 65%, 75%, 85%, 95%, or 98%) identical to: (a) a nucleicacid molecule that encodes the polypeptide of SEQ ID NO:1, 3, or 5; (b)the nucleotide sequence of SEQ ID NO:2, 4, or 6; or (c) a nucleic acidmolecule which includes: (i) a segment of at least 30 (e.g., at least50, 60, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400,425, 450, 500, 550, 600, 650, 700, 800, 900, or 940) nucleotides of SEQID NO:2 or SEQ ID NO:4; or (ii) a segment of at least 30 (e.g., at least50, 60, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400,425, 450, 500, 550, 600, 650, 700, 800, or 840 nucleotides of SEQ IDNO:6.

The determination of percent identity between two sequences isaccomplished using the mathematical algorithm of Karlin and Altschul,Proc. Natl. Acad. Sci. USA 90, 5873-5877, 1993. Such an algorithm isincorporated into the BLASTN and BLASTP programs of Altschul et al.(1990) J. Mol. Biol. 215, 403-410. BLAST nucleotide searches areperformed with the BLASTN program, score=100, wordlength=12 to obtainnucleotide sequences homologous to B7-H3- or B7-H4-encoding nucleicacids. BLAST protein searches are performed with the BLASTP program,score=50, wordlength=3 to obtain amino acid sequences homologous toB7-H3 or B7-H4. To obtain gapped alignments for comparative purposes,Gapped BLAST is utilized as described in Altschul et al. (1997) NucleicAcids Res. 25, 3389-3402. When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) are used (See http://www.ncbi.nlm.nih.gov).

Hybridization can also be used as a measure of homology between twonucleic acid sequences. A B7-H3- or B7-H4-encoding nucleic acidsequence, or a portion thereof, can be used as hybridization probeaccording to standard hybridization techniques. The hybridization of aB7-H3 probe to DNA from a test source (e.g., a mammalian cell) is anindication of the presence of B7-H3 DNA in the test source and thehybridization of a B7-H4 probe to DNA from a test source (e.g., amammalian cell) is an indication of the presence of B7-H4 DNA in thetest source. Hybridization conditions are known to those skilled in theart and can be found in Current Protocols in Molecular Biology, JohnWiley & Sons, N.Y., 6.3.1-6.3.6, 1991. Moderate hybridization conditionsare defined as equivalent to hybridization in 2× sodium chloride/sodiumcitrate (SSC) at 30° C., followed by one or more washes in 1×SSC, 0.1%SDS at 50-60° C. Highly stringent conditions are defined as equivalentto hybridization in 6× sodium chloride/sodium citrate (SSC) at 45° C.,followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65° C.

The invention also encompasses: (a) vectors that contain any of theforegoing B7-H3- and B7-H4-related coding sequences and/or theircomplements (that is, “antisense” sequence); (b) expression vectors thatcontain any of the foregoing B7-H3-related and B7-H4-related codingsequences operatively associated with any transcriptional/translationalregulatory elements (examples of which are given below) necessary todirect expression of the coding sequences; (c) expression vectorscontaining, in addition to sequences encoding a B7-H3 or a B7-H4polypeptide, nucleic acid sequences that are unrelated to nucleic acidsequences encoding B7-H3 or a B7-H4, such as molecules encoding areporter, marker, or a signal peptide, e.g., fused to B7-H3 or B7-H4;and (d) genetically engineered host cells that contain any of theforegoing expression vectors and thereby express the nucleic acidmolecules of the invention.

Recombinant nucleic acid molecules can contain a sequence encoding B7-H3or B7-H4, B7-H3 having a heterologous signal sequence, or B7-H4 havingan heterologous signal sequence. The full length B7-H3 polypeptide, adomain of B7-H3, or a fragment thereof may be fused to additionalpolypeptides, as described below. In addition, the full-length B7-H4polypeptide, a domain of B7-H4, or a fragment thereof may be fused toadditional polypeptides, as described below. Similarly, the nucleic acidmolecules of the invention can encode the mature form of B7-H3 or B7-H4or a form that includes an exogenous polypeptide which facilitatessecretion.

The transcriptional/translational regulatory elements referred to aboveand which are further described below, include, but are not limited to,inducible and non-inducible promoters, enhancers, operators and otherelements, which are known to those skilled in the art, and which driveor otherwise regulate gene expression. Such regulatory elements includebut are not limited to the cytomegalovirus hCMV immediate early gene,the early or late promoters of SV40 adenovirus, the lac system, the trpsystem, the TAC system, the TRC system, the major operator and promoterregions of phage A, the control regions of fd coat protein, the promoterfor 3-phosphoglycerate kinase, the promoters of acid phosphatase, andthe promoters of the yeast α-mating factors.

Similarly, the nucleic acid can form part of a hybrid gene encodingadditional polypeptide sequences, for example, sequences that functionas a marker or reporter. Examples of marker or reporter genes includeβ-lactamase, chloramphenicol acetyltransferase (CAT), adenosinedeaminase (ADA), aminoglycoside phosphotransferase (neo^(r), G418^(r)),dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH),thymidine kinase (TK), lacZ (encoding β-galactosidase), and xanthineguanine phosphoribosyltransferase (XGPRT). As with many of the standardprocedures associated with the practice of the invention, skilledartisans will be aware of additional useful reagents, for example,additional sequences that can serve the function of a marker orreporter. Generally, the hybrid polypeptide will include a first portionand a second portion; the first portion being a B7-H3 or B7-H4polypeptide (or a fragment of such a polypeptide) and the second portionbeing, for example, the reporter described above or an Ig constantregion or part of an Ig constant region, e.g., the CH2 and CH3 domainsof IgG2a.

The expression systems that may be used for purposes of the inventioninclude, but are not limited to, microorganisms such as bacteria (forexample, E. coli and B. subtilis) transformed with recombinantbacteriophage DNA, plasmid DNA, or cosmid DNA expression vectorscontaining the nucleic acid molecules of the invention; yeast (forexample, Saccharomyces and Pichia) transformed with recombinant yeastexpression vectors containing the nucleic acid molecules of theinvention, preferably containing the nucleic acid sequence encodingB7-H3 (SEQ ID NO:2 or 4) or B7-H4 (SEQ ID NO:6); insect cell systemsinfected with recombinant virus expression vectors (for example,baculovirus) containing the nucleic acid molecules of the invention;plant cell systems infected with recombinant virus expression vectors(for example, cauliflower mosaic virus (CaMV) and tobacco mosaic virus(TMV)) or transformed with recombinant plasmid expression vectors (forexample, Ti plasmid) containing B7-H3 or B7-H4 nucleotide sequences; ormammalian cell systems (for example, COS, CHO, BHK, 293, VERO, HeLa,MDCK, WI38, and NIH 3T3 cells) harboring recombinant expressionconstructs containing promoters derived from the genome of mammaliancells (for example, the metallothionein promoter) or from mammalianviruses (for example, the adenovirus late promoter and the vacciniavirus 7.5K promoter). Also useful as host cells are primary or secondarycells obtained directly from a mammal transfected with a plasmid vectoror infected with a viral vector.

Polypeptides and Polypeptide Fragments

The polypeptides of the invention include B7-H3, B7-H4, and functionalfragments of these polypeptides. The polypeptides embraced by theinvention also include fusion proteins which contain either full-lengthB7-H3 or B7-H4 or a functional fragment of either polypeptide fused toan unrelated amino acid sequence. The unrelated sequences can beadditional functional domains or signal peptides. Signal peptides aredescribed in greater detail and exemplified below. The polypeptides canalso be any of those described above but with one or more conservativesubstitutions.

The polypeptides can be purified from natural sources (e.g., blood,serum plasma, tissues or cells such as T cells or any cell thatnaturally produces B7-H3 or B7-H4). Smaller peptides (less than 50 aminoacids long) can also be conveniently synthesized by standard chemicalmeans. In addition, both polypeptides and peptides can be produced bystandard in vitro recombinant DNA techniques and in vivorecombination/genetic recombination (e.g., transgenesis), using thenucleotide sequences encoding the appropriate polypeptides or peptides.Methods well known to those skilled in the art can be used to constructexpression vectors containing relevant coding sequences and appropriatetranscriptional/translational control signals. See, for example, thetechniques described in Sambrook et al., Molecular Cloning: A LaboratoryManual (2nd Ed.) [Cold Spring Harbor Laboratory, N.Y., 1989], andAusubel et al., Current Protocols in Molecular Biology, [GreenPublishing Associates and Wiley Interscience, N.Y., 1989].

Polypeptides and fragments of the invention also include those describedabove, but modified for in vivo use by the addition, the amino- and/orcarboxyl-terminal ends, of a blocking agent to facilitate survival ofthe relevant polypeptide in vivo. This can be useful in those situationsin which the peptide termini tend to be degraded by proteases prior tocellular uptake. Such blocking agents can include, without limitation,additional related or unrelated peptide sequences that can be attachedto the amino and/or carboxyl terminal residues of the peptide to beadministered. This can be done either chemically during the synthesis ofthe peptide or by recombinant DNA technology by methods familiar toartisans of average skill.

Alternatively, blocking agents such as pyroglutamic acid or othermolecules known in the art can be attached to the amino and/or carboxylterminal residues, or the amino group at the amino terminus or carboxylgroup at the carboxyl terminus can be replaced with a different moiety.Likewise, the peptides can be covalently or noncovalently coupled topharmaceutically acceptable “carrier” proteins prior to administration.

Also of interest are peptidomimetic compounds that are designed basedupon the amino acid sequences of the functional peptide fragments.Peptidomimetic compounds are synthetic compounds having athree-dimensional conformation (i.e., a “peptide motif”) that issubstantially the same as the three-dimensional conformation of aselected peptide. The peptide motif provides the peptidomimetic compoundwith the ability to co-stimulate T cells in a manner qualitativelyidentical to that of the B7-H3 or B7-H4 functional peptide fragment fromwhich the peptidomimetic was derived. Peptidomimetic compounds can haveadditional characteristics that enhance their therapeutic utility, suchas increased cell permeability and prolonged biological half-life.

The peptidomimetics typically have a backbone that is partially orcompletely non-peptide, but with side groups that are identical to theside groups of the amino acid residues that occur in the peptide onwhich the peptidomimetic is based. Several types of chemical bonds,e.g., ester, thioester, thioamide, retroamide, reduced carbonyl,dimethylene and ketomethylene bonds, are known in the art to begenerally useful substitutes for peptide bonds in the construction ofprotease-resistant peptidomimetics.

Methods of Co-Stimulating a T Cell

The methods of the invention involve contacting a T cell with a B7-H3 orB7-H4 polypeptide of the invention, or a functional fragment thereof, inorder to co-stimulate the T cell. Such polypeptides or functionalfragments can have amino acid sequences identical to wild-type sequencesor they can contain one or more conservative substitutions. Thecontacting can occur before, during, or after activation of the T cell.Contacting of the T cell with the B7-H3 or B7-H4 polypeptide willpreferably be at substantially the same time as activation. Activationcan be, for example, by exposing the T cell to an antibody that binds tothe TCR or one of the polypeptides of the CD3 complex that is physicallyassociated with the TCR. Alternatively, the T cell can be exposed toeither an alloantigen (e.g., a MHC alloantigen) on, for example, anantigen presenting cell (APC) (e.g., a dendritic cell, a macrophage, amonocyte, or a B cell) or an antigenic peptide produced by processing ofa protein antigen by any of the above APC and presented to the T cell byMHC molecules on the surface of the APC. The T cell can be a CD4+ T cellor a CD8+ T cell. The B7-H3 or B7-H4 polypeptide can be added to thesolution containing the cells, or it can be expressed on the surface ofan APC, e.g., an APC presenting an alloantigen or an antigen peptidebound to an MHC molecule. Alternatively, if the activation is in vitro,the B7-H3 or B7-H4 polypeptide can be bound to the floor of a therelevant culture vessel, e.g., a well of a plastic microtiter plate.

The methods can be performed in vitro, in vivo, or ex vivo. In vitroapplication of B7-H3 or B7-H4 can be useful, for example, in basicscientific studies of immune mechanisms or for production of activated Tcells for use in either studies on T cell function or, for example,passive immunotherapy. Furthermore, B7-H3 or B7-H4 could be added to invitro assays (e.g., in T cell proliferation assays) designed to test forimmunity to an antigen of interest in a subject from which the T cellswere obtained. Addition of B7-H3 or B7-H4 to such assays would beexpected to result in a more potent, and therefore more readilydetectable, in vitro response. However, the methods of the inventionwill preferably be in vivo or ex vivo (see below).

The B7-H3 and B7-H4 proteins and variants thereof are generally usefulas immune response-stimulating therapeutics. For example, thepolypeptides of the invention can be used for treatment of diseaseconditions characterized by immunosuppression: e.g., cancer, AIDS orAIDS-related complex, other virally or environmentally-inducedconditions, and certain congenital immune deficiencies. The polypeptidesmay also be employed to increase immune function that has been impairedby the use of radiotherapy of immunosuppressive drugs such as certainchemotherapeutic agents, and therefore are particularly useful whengiven in conjunction with such drugs or radiotherapy. The polypeptidescan, furthermore, be used to enhance immune responses in normalsubjects.

These methods of the invention can be applied to a wide range ofspecies, e.g., humans, non-human primates, horses, cattle, pigs, sheep,goats, dogs, cats, rabbits, guinea pigs, hamsters, rats, and mice.

In Vivo Approaches

In one in vivo approach, a B7-H3 or B7-H4 polypeptide (or a functionalfragment thereof) itself is administered to the subject. Generally, thecompounds of the invention will be suspended in apharmaceutically-acceptable carrier (e.g., physiological saline) andadministered orally or by intravenous infusion, or injectedsubcutaneously, intramuscularly, intraperitoneally, intrarectally,intravaginally, intranasally, intragastrically, intratracheally, orintrapulmonarily. They are preferably delivered directly to anappropriate lymphoid tissue (e.g. spleen, lymph node, ormucosal-associated lymphoid tissue (MALT)). The dosage required dependson the choice of the route of administration, the nature of theformulation, the nature of the patient's illness, the subject's size,weight, surface area, age, and sex, other drugs being administered, andthe judgment of the attending physician. Suitable dosages are in therange of 0.01-100.0 μg/kg. Wide variations in the needed dosage are tobe expected in view of the variety of polypeptides and fragmentsavailable and the differing efficiencies of various routes ofadministration. For example, oral administration would be expected torequire higher dosages than administration by i.v. injection. Variationsin these dosage levels can be adjusted using standard empirical routinesfor optimization as is well understood in the art. Administrations canbe single or multiple (e.g., 2- or 3-, 4-, 6-, 8-, 10-, 20-, 50-, 100-,150-, or more fold). Encapsulation of the polypeptide in a suitabledelivery vehicle (e.g., polymeric microparticles or implantable devices)may increase the efficiency of delivery, particularly for oral delivery.

Alternatively, a polynucleotide containing a nucleic acid sequenceencoding the B7-H3 or B7-H4 polypeptide or functional fragment thereofcan be delivered to an appropriate cell of the animal. Expression of thecoding sequence will preferably be directed to lymphoid tissue of thesubject by, for example, delivery of the polynucleotide to the lymphoidtissue. This can be achieved by, for example, the use of a polymeric,biodegradable microparticle or microcapsule delivery vehicle, sized tooptimize phagocytosis by phagocytic cells such as macrophages. Forexample, PLGA (poly-lacto-co-glycolide) microparticles approximately1-10 μm in diameter can be used. The polynucleotide is encapsulated inthese microparticles, which are taken up by macrophages and graduallybiodegraded within the cell, thereby releasing the polynucleotide. Oncereleased, the DNA is expressed within the cell. A second type ofmicroparticle is intended not to be taken up directly by cells, butrather to serve primarily as a slow-release reservoir of nucleic acidthat is taken up by cells only upon release from the micro-particlethrough biodegradation. These polymeric particles should therefore belarge enough to preclude phagocytosis i.e., larger than 5 μm andpreferably larger than 20 μm.

Another way to achieve uptake of the nucleic acid is using liposomes,prepared by standard methods. The vectors can be incorporated alone intothese delivery vehicles or co-incorporated with tissue-specificantibodies. Alternatively, one can prepare a molecular conjugatecomposed of a plasmid or other vector attached to poly-L-lysine byelectrostatic or covalent forces. Poly-L-lysine binds to a ligand thatcan bind to a receptor on target cells [Cristiano et al. (1995), J. Mol.Med. 73, 479]. Alternatively, lymphoid tissue specific targeting can beachieved by the use of lymphoid tissue-specific transcriptionalregulatory elements (TRE) such as a B lymphocyte, T lymphocyte, ordendritic cell specific TRE. Lymphoid tissue specific TRE are known[Thompson et al. (1992), Mol. Cell. Biol. 12, 1043-1053; Todd et al.(1993), J. Exp. Med. 177, 1663-1674; Penix et al. (1993), J. Exp. Med.178, 1483-1496]. Delivery of “naked DNA” (i.e., without a deliveryvehicle) to an intramuscular, intradermal, or subcutaneous site, isanother means to achieve in vivo expression.

In the relevant polynucleotides (e.g., expression vectors) the nucleicacid sequence encoding the B7-H3 or B7-H4 polypeptide or functionalfragment of interest with an initiator methionine and optionally atargeting sequence is operatively linked to a promoter orenhancer-promoter combination.

Short amino acid sequences can act as signals to direct proteins tospecific intracellular compartments. For example, hydrophobic signalpeptides (e.g., MAISGVPVLGFFIIAVLMSAQESWA (SEQ ID NO:7)) are found atthe amino terminus of proteins destined for the ER. While the sequenceKFERQ (SEQ ID NO:8) (and other closely related sequences) is known totarget intracellular polypeptides to lysosomes, other sequences (e.g.,MDDQRDLISNNEQLP (SEQ ID NO:9) direct polypeptides to endosomes. Inaddition, the peptide sequence KDEL (SEQ ID NO:10) has been shown to actas a retention signal for the ER. Each of these signal peptides, or acombination thereof, can be used to traffic the B7-H3 or B7-H4polypeptides or functional fragments of the invention as desired. DNAsencoding the B7-H3 or B7-H4 polypeptides or functional fragmentscontaining targeting signals will be generated by PCR or other standardgenetic engineering or synthetic techniques.

A promoter is a TRE composed of a region of a DNA molecule, typicallywithin 100 basepairs upstream of the point at which transcriptionstarts. Enhancers provide expression specificity in terms of time,location, and level. Unlike a promoter, an enhancer can function whenlocated at variable distances from the transcription site, provided apromoter is present. An enhancer can also be located downstream of thetranscription initiation site. To bring a coding sequence under thecontrol of a promoter, it is necessary to position the translationinitiation site of the translational reading frame of the peptide orpolypeptide between one and about fifty nucleotides downstream (3′) ofthe promoter. The coding sequence of the expression vector isoperatively linked to a transcription terminating region.

Suitable expression vectors include plasmids and viral vectors such asherpes viruses, retroviruses, vaccinia viruses, attenuated vacciniaviruses, canary pox viruses, adenoviruses and adeno-associated viruses,among others.

Polynucleotides can be administered in a pharmaceutically acceptablecarrier. Pharmaceutically acceptable carriers are biologicallycompatible vehicles which are suitable for administration to a human,e.g., physiological saline. A therapeutically effective amount is anamount of the polynucleotide which is capable of producing a medicallydesirable result (e.g., an enhanced T cell response) in a treatedanimal. As is well known in the medical arts, the dosage for any onepatient depends upon many factors, including the patient's size, bodysurface area, age, the particular compound to be administered, sex, timeand route of administration, general health, and other drugs beingadministered concurrently. Dosages will vary, but a preferred dosage foradministration of polynucleotide is from approximately 10⁶ to 10¹²copies of the polynucleotide molecule. This dose can be repeatedlyadministered, as needed. Routes of administration can be any of thoselisted above.

Included in these in vivo approaches, are methods of co-stimulating a Tcell that involve administering more than one co-stimulatory molecule orfunctional fragment thereof. Such combinations can be any combination ofone or more of co-stimulatory polypeptides, e.g., B7-1, B7-2, B7-H1,B7-H2, B7-H3, B7-H4, 4-1BB, OX40, or HVEM and functional fragments ofany of these. The proteins or functional fragments per se can beadministered (as above) or nucleic acids (e.g., expression vectors)encoding the proteins or functional fragments can be administered (asabove). Where expression vectors are used, a single vector containingcoding sequences for two or more of the co-stimulatory polypeptides orfunctional fragments can be administered. Alternatively, multiple (e.g.,2, 3, 4, 5, or 6) individual vectors, each encoding one or more (e.g.,2, 3, 4, 5, or 6) of the co-stimulatory polypeptides or functionalfragments thereof can be administered.

Ex Vivo Approaches

Peripheral blood mononuclear cells (PBMC) can be withdrawn from thepatient or a suitable donor and exposed ex vivo to an activatingstimulus (see above) and a B7-H3 or B7-H4 polypeptide or polypeptidefragment (whether in soluble form or attached to a sold support bystandard methodologies). The PBMC containing highly activated T cellsare then introduced into the same or a different patient.

An alternative ex vivo strategy can involve transfecting or transducingcells obtained from the subject with a polynucleotide encoding a B7-H3or B7-H4 polypeptide or functional fragment-encoding nucleic acidsequences described above. The transfected or transduced cells are thenreturned to the subject. While such cells would preferably behemopoietic cells (e.g., bone marrow cells, macrophages, monocytes,dendritic cells, or B cells) they could also be any of a wide range oftypes including, without limitation, fibroblasts, epithelial cells,endothelial cells, keratinocytes, or muscle cells in which they act as asource of the B7-H3 or B7-H4 polypeptide or functional fragments thereoffor as long as they survive in the subject. The use of hemopoieticcells, that include the above APC, would be particularly advantageous inthat such cells would be expected to home to, among others, lymphoidtissue (e.g., lymph nodes or spleen) and thus the B7-H3 or B7-H4polypeptide or functional fragment would be produced in highconcentration at the site where they exert their effect, i.e.,enhancement of an immune response. In addition, if APC are used, the APCexpressing the exogenous B7-H3 or B7-H4 molecule can be the same APCthat presents an alloantigen or antigenic peptide to the relevant Tcell. The B7-H3 or B7-H4 polypeptides can be secreted by the APC orexpressed on its surface. Prior to returning the recombinant APC to thepatient, they can optionally be exposed to sources of antigens orantigenic peptides of interest, e.g., those of tumors, infectiousmicroorganisms, or autoantigens. The same genetic constructs andtrafficking sequences described for the in vivo approach can be used forthis ex vivo strategy. Furthermore, tumor cells, preferably obtainedfrom a patient, can be transfected or transformed by a vector encoding aB7-H3 or B7-H4 polypeptide or functional fragment thereof. The tumorcells, preferably treated with an agent (e.g., ionizing irradiation)that ablates their proliferative capacity, are then returned to thepatient where, due to their expression of the exogenous B7-H3 or B7-H4(on their cell surface or by secretion), they can stimulate enhancedtumoricidal T cell immune responses. It is understood that the tumorcells which, after transfection or transformation, are injected into thepatient, can also have been originally obtained from an individual otherthan the patient.

The ex vivo methods include the steps of harvesting cells from asubject, culturing the cells, transducing them with an expressionvector, and maintaining the cells under conditions suitable forexpression of the B7-H3 or B7-H4 polypeptide or functional fragment.These methods are known in the art of molecular biology. Thetransduction step is accomplished by any standard means used for ex vivogene therapy, including calcium phosphate, lipofection, electroporation,viral infection, and biolistic gene transfer. Alternatively, liposomesor polymeric microparticles can be used. Cells that have beensuccessfully transduced are then selected, for example, for expressionof the coding sequence or of a drug resistance gene. The cells may thenbe lethally irradiated (if desired) and injected or implanted into thepatient.

It is understood that in these ex vivo procedures, the cells to beintroduced into a subject can be transfected or transformed with one ormore (e.g., two, three, four, five, or six) expression vectorscontaining one or more (e.g., two, three, four, five, or six) sequencesencoding any of the co-stimulatory molecules listed above (e.g., B7-1,B7-2, B7-H1, B7-H2, B7-H3, or B7-H4) or functional fragments thereofprior to introduction.

Methods of Screening for Compounds that Inhibit or Enhance ImmuneResponses.

The invention provides methods for testing compounds (small molecules ormacromolecules) that inhibit or enhance an immune response. Such amethod can involve, e.g., culturing a B7-H3 or B7-H4 polypeptide of theinvention (or a functional fragment thereof) with T cells in thepresence of a T cell stimulus (see above). The B7-H3 or B7-H4 moleculecan be in solution or membrane bound (e.g., expressed on the surface ofthe T cells) and it can be natural or recombinant. Furthermore, theB7-H3 or B7-H4 polypeptides (or functional fragments thereof) can haveamino acid sequences identical to wild-type sequences or they can haveone or more conservative substitutions. Compounds that inhibit the Tcell response will likely be compounds that inhibit an immune responsewhile those that enhance the T cell response will likely be compoundsthat enhance an immune response.

The invention also relates to using B7-H3 or B7-H4 or functionalfragments thereof to screen for immunomodulatory compounds that caninteract with B7-H3 or B7-H4. One of skill in the art would know how touse standard molecular modeling or other techniques to identify smallmolecules that would bind to T cell interactive sites of B7-H3 or B7-H4.One such example is provided in Broughton (1997) Curr. Opin. Chem. Biol.1, 392-398.

A candidate compound whose presence requires at least 1.5-fold (e.g.,2-fold, 4-fold, 6-fold, 10-fold, 150-fold, 1000-fold, 10.000-fold, or100.000-fold) more B7-H3 or B7-H4 in order to achieve a definedarbitrary level of T cell activation than in the absence of the compoundcan be useful for inhibiting an immune response. On the other hand, acandidate compound whose presence requires at least 1.5 fold (e.g.,2-fold, 4-fold, 6-fold, 10-fold, 100-fold, 1000-fold, 10,000 fold, or100.000-fold) less B7-H3 or B7-H4 to achieve a defined arbitrary levelof T cell activation than in the absence of the compound can be usefulfor enhancing an immune response. Compounds capable of interfering withor modulating B7-H3 or B7-H4 function are good candidates forimmunosuppressive immunoregulatory agents, e.g., to modulate anautoimmune response or suppress allogeneic or xenogeneic graftrejection.

B7-H3 and B7-H4 Antibodies

The invention features antibodies that bind to the B7-H3 or B7-H4polypeptides or fragments of such polypeptides. Such antibodies can bepolyclonal antibodies present in the serum or plasma of animals (e.g.,mice, rabbits, rats, guinea pigs, sheep, horses, goats, cows, or pigs)which have been immunized with the relevant B7-H3 or B7-H4 polypeptideor peptide fragment using methods, and optionally adjuvants, known inthe art. Such polyclonal antibodies can be isolated from serum or plasmaby methods known in the art. Monoclonal antibodies that bind to theabove polypeptides or fragments are also embodied by the invention.Methods of making and screening monoclonal antibodies are well known inthe art.

Once the desired antibody-producing hybridoma has been selected andcloned, the resultant antibody can be produced in a number of methodsknown in the art. For example, the hybridoma can be cultured in vitro ina suitable medium for a suitable length of time, followed by therecovery of the desired antibody from the supernatant. The length oftime and medium are known or can be readily determined.

Additionally, recombinant antibodies specific for B7-H3 or B7-H4, suchas chimeric and humanized monoclonal antibodies comprising both humanand non-human portions, are within the scope of the invention. Suchchimeric and humanized monoclonal antibodies can be produced byrecombinant DNA techniques known in the art, for example, using methodsdescribed in Robinson et al., International Patent PublicationPCT/US86/02269; Akira et al., European Patent Application 184,187;Taniguchi, European Patent Application 171,496; Morrison et al.,European Patent Application 173,494; Neuberger et al., PCT ApplicationWO 86/01533; Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al.,European Patent Application 125,023; Better et al. (1988) Science 240,1041-43; Liu et al. (1987) J. Immunol. 139, 3521-26; Sun et al. (1987)PNAS 84, 214-18; Nishimura et al. (1987) Canc. Res. 47, 999-1005; Woodet al. (1985) Nature 314, 446-49; Shaw et al. (1988) J. Natl. CancerInst. 80, 1553-59; Morrison, (1985) Science 229, 1202-07; Oi et al.(1986) BioTechniques 4, 214; Winter, U.S. Pat. No. 5,225,539; Jones etal. (1986) Nature 321, 552-25; Veroeyan et al. (1988) Science 239, 1534;and Beidler et al. (1988) J. Immunol. 141, 4053-60.

Also included within the scope of the invention are antibody fragmentsand derivatives which contain at least the functional portion of theantigen binding domain of an antibody that binds specifically to B7-H3or B7-H4. Antibody fragments that contain the binding domain of themolecule can be generated by known techniques. For example, suchfragments include, but are not limited to: F(ab′)₂ fragments which canbe produced by pepsin digestion of antibody molecules; Fab fragmentswhich can be generated by reducing the disulfide bridges of F(ab′)₂fragments; and Fab fragments which can be generated by treating antibodymolecules with papain and a reducing agent. See, e.g., NationalInstitutes of Health, 1 Current Protocols In Immunology, Coligan et al.,ed. 2.8, 2.10 (Wiley Interscience, 1991). Antibody fragments alsoinclude Fv (e.g., single chain Fv (scFv)) fragments, i.e., antibodyproducts in which there are no constant region amino acid residues. Suchfragments can be produced, for example, as described in U.S. Pat. No.4,642,334 which is incorporated herein by reference in its entirety.

The following examples are meant to illustrate, not limit, theinvention.

Example 1 Materials and Methods

Cell lines, cell preparation and activation. The adenovirus-transformedhuman kidney epithelial line 293 and the histocytic lymphoma line U937were purchased from ATCC (Manassas, Va.). The 624mel melanoma line was agift from Dr. Rong-Fu Wang (Surgery Branch, NCI). Cell lines were grownand maintained in DMEM or RPMI 1640 media (GIBCO, Gaithersburg, Md.)supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, Utah),25 mM HEPES (Life Technologies, Grand Island, USA), 2 mM L-glutamine(Life Technologies), 100 U/ml penicillin (Life Technologies), 100 μg/mlstreptomycin (Life Technologies), and 5×10⁻⁵M 2-mercaptoethanol (VWR,Chicago, Ill.). Methods for the preparation of peripheral bloodmononuclear cells (PBMC) from healthy donors and monocyte-deriveddendritic cells (DC) were described previously [Chapoval et al. (2000)Blood. 95, 2346-2351]. Fluorescence flow cytometry (FFC) analysis usingmonoclonal antibodies (mAb) specific for B7-1, B7-2, and MHC class IIindicated that >99% of cells in the monocyte-derived DC preparations hada typical DC phenotype. Total T cell-enriched populations were obtainedby passing nonadherent PBMC through nylon wool columns as describedpreviously [Tamada et al. (2000) J. Immunol. 164, 4105-4110]. CD4⁺ andCD8⁺ T cell subsets were further purified using the MACS magnetic beadsystem (Miltenyl Biotec, Auburn, Calif.) according to the manufacturer'sinstructions. The purity of the T cell subset preparations were >98%based on FFC analysis using T cell subset-specific mAb. For theactivation of U937 cells or DC, the cells (1×10⁶ cells/ml) were culturedfor 24 h with lipopolysaccharide (LPS; 1 μg/ml). Adherent PBMC wereactivated with a combination of LPS (100 ng/ml) and human interferon-γ(IFN-γ; 1500 IU/ml; R&D Systems, Minneapolis Minn.). For activation of Tand B cells, PBMC (2.5×10⁶ cells/ml) were cultured for 24 h withphytohemagglutinin (PHA; (5 μg/ml)) or LPS (1 μg/ml), respectively. Allcell types were also activated with phorbol myristic acid (PMA; 5 ng/ml)and ionomycin (250 ng/ml).

Cloning and sequencing of full-length human B7-H3 cDNA. The NationalCenter for Biotechnology Information (NCBI and Human Genome Sciences,Inc. expressed sequence tag (EST) databases containing the sequencesof >500 different cDNA libraries were screened for cDNA sequences havinghomology to published B7-1, B7-2, B7-H1, and B7-H2 sequences using theBLASTN and TBLASTN algorithms. Information obtained from this screeningwas used to generate appropriate PCR primers and full-length human cDNAmolecules encoding B7-H3 was generated by PCR from a THP-1 cDNA libraryprepared by the SMART PCR cDNA synthesis kit (Clontech, Palo Alto,Calif.). The resulting PCR product was cloned into the pcDNA3.1(−)vector (Invitrogen, Carlsbad, Calif.). The nucleotide sequence of theB7-H3 encoding cDNA molecules was verified by sequencing of severalindependent clones. For transient expression of the B7-H3 gene, thevector containing full-length B7-H3.1 encoding cDNA (pcDNA/B7-H3) orcontrol parental vector (pcDNA3.1(−)) was transfected into 293 or 624melcells by Fugene 6 (Boeheringer-Mannheim) according to the manufacturer'sinstructions.

Production of fusion proteins. Recombinant B7-H3Ig fusion protein wasprepared by fusing the coding region of the extracellular domain ofB7-H3 to the Fc constant region of mouse IgG2a as described previously[Chapoval et al. (2000). Methods Mol. Med. 45:247-255]. An expressionvector containing a DNA sequence encoding B7-H3Ig was transfected into293 cells by calcium phosphate precipitation and cultured in serum-freeDMEM. The supernatant was collected at 72 h and the fusion protein waspurified by Protein G sepharose columns (Pharmacia, Uppsala, Sweden).The purity and expected molecular weight of the fusion protein wereconfirmed by electrophoresis on polyacrylamide gels and by Western blotusing the anti-B7-H3 antibody described below. Analogous fusion proteins(ICOSIg, B7-1Ig, and CTLA4Ig) containing the extracellular domains ofICOS, B7-1, and CTLA4, respectively, were prepared by a similar method.

RNA analysis. The expression of B7-H3 mRNA in human tissues was analyzedusing human multiple-tissue and tumor line Northern blots purchased fromClontech (Palo Alto, Calif.). Full-length cDNA encoding B7-H3 waslabeled with [³²P]-dCTP and hybridized to filter membranes according tothe manufacturer's protocol.

Immunization, antibody production and FACS analysis. For production ofantibody specific for human B7-H3, a peptide consisting of amino acids142-165 (YSKPSMTLEPNKDLRPGDTVTITC) (SEQ ID NO:12) spanning a hydrophilicregion of B7-H3 was synthesized and conjugated to KLH as describedpreviously [Tamada et al. (2000) J. Immunol. 164, 4105-4110]. Polyclonalantibodies were prepared by immunizing BALB/c mice with the peptide incomplete Freund's adjuvant (Sigma) and subsequently boosting two timeswith the peptides in incomplete Freund's adjuvant. Blood was collectedand sera prepared 10 days after the final boost. The specificity of theantiserum was determined by ELISA against B7-H3Ig and indirectimmunofluorescence of 293 cells transfected to express B7-H3, B7-H2,B7-H1, and B7-1. Serum prepared from the blood of non-immunized BALB/cmice was used as a negative control. Substantially identical resultswere obtained with two antisera generated in the same way byimmunization of mice with B7-H3 hydrophilic region peptidescorresponding to amino acid residues 166-189 (SSYRGYPEAEVFWQDGQGVPLTGN)(SEQ ID NO:13) of B7-H3.1 or amino acid residues 223-247(RNPVLQQDAHGSVTITGQPMTFPPE) (SEQ ID NO:14) of B7-H3.

For analysis of B7-H3 expression and B7-H3 interaction with a putativecounter-receptor, cells were incubated either with anti-B7-H3 antibodies(1:1000 dilution in PBS), CTLA4Ig (5 μg/sample) or ICOSIg (5 μg/sample)on ice. After a 45 min incubation, cells were washed and cultured for 45min with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgF(ab′)₂ (BioSource, Camarillo, Calif.). For two color staining, cellswere washed and further incubated with phycoerythrin (PE)-conjugated mAbspecific for CD3, CD14, or CD19 (PharMingen, San Diego, Calif.). For theanalysis of CD3+, CD19+, and CD14+ cells, peripheral blood mononuclearcells (PBMC) (untreated or activated as described below) were stainedwith anti-B3-H7 antiserum (green fluorescence) and either anti-CD3,anti-CD19, or anti-CD14 monoclonal antibody (mAb) (orange fluorescence).The proportion of B7-H3 expressing cells (green fluorescence) within theCD3+, the CD19+, and the CD14+ populations was assessed by gating theflow cytometer on the relevant populations (orange fluorescence).Expression of a B7-H3 counter-receptor was analyzed by incubation ofcells with B7-H3Ig (5 μg/sample) with subsequent staining usingFITC-conjugated goat anti-mouse Ig F(ab′)₂. Single or double stainedcells were analyzed using the Becton Dickinson FACScan (Mountain View,Calif.).

T cell proliferation an cytokine and cytokine production assay. T cellproliferation was measured as previously described [Dong et al. (1999)Nat. Med 5, 1365-1369]. Briefly, flat-bottom 96-well microtiter cultureplates were coated at 4° C. overnight with 50 μl/well of anti-CD3 mAb(40 ng/ml) and subsequently coated with the indicated concentrations ofB7-H3Ig, B7-1Ig, or control Ig at 37° C. for 4 h. T cells were added tothe wells at the indicated concentrations. For measurements of T cellproliferation, the plates were cultured for 72 h and [³H]-thymidine (1μCi/well) was added 18 h prior to harvesting of the cultures.[³H]-thymidine incorporation was measured with a MicroBeta Trilix liquidscintillation counter (Wallac, Turku, Finland). For measurements ofcytokine (interleukin- (IL-)2, IL-10, and interferon-γ (IFN-γ)production, supernatants were removed from the cultures at the indicatedtimes after culture initiation and subjected to ELISA for the cytokinesusing commercially available ELISA kits (PharMingen).

CTL generation and cytotoxicity assay. CTL were generated in 24-wellculture plates by co-culturing of nylon wool column-purified T cells(5×10⁶ cells/well) in the presence of irradiated (10,000 rad) 624melcells (1×10⁵ cells/well) transfected with either pcDNA/B7-H3 or controlparent pcDNA3.1 (−) plasmid. The cultured cells were harvested on day 5and CTL activity against parental 624mel was measured in a standard 4 h⁵¹Cr-release assay [Chapoval et al. (1998) J. Immunol. 161, 6977-6984].

Cytokine gene expression. Total RNA was prepared using TRI Reagent(Sigma) from 5×10⁶ T cells which had been cultured in 24-well tissueculture plates for 72 h with a suboptimal dose of anti-CD3 antibody(coated onto tissue culture plate wells at a concentration of 200 ng/ml)and with either control Ig (coated onto tissue culture plate wells at aconcentration of 5 ug/ml), B7-1Ig (coated onto tissue culture platewells at a concentration of 5 ug/ml), or B7-H3Ig (coated onto tissueculture plate wells at a concentration of 5 ug/ml). Ten micrograms ofthe total RNA from each of the three cell populations were used as atemplate to produce ³²P labeled cDNA by standard procedures usingretroviral (MMTV) reverse transcriptase (Promega, Madison, Wis.) and[α-³²P]-dCTP (NEN, Boston, Mass.). The Human Common Cytokine-1 GEArrayKit (No hGEA9912099, SuperArray Inc. Bethesda, Md.) was used todetermine the relative amounts of cDNA encoding 23 different cytokines(see FIG. 14) in the three ³²P labeled cDNA samples as an indirectmeasure of the of the relative amounts of corresponding mRNA in thethree cell populations. The kit includes cytokine mRNA arrays which aremembranes containing mRNA encoding the 23 cytokines and two housekeeping protein, beta-actin and glyceraldehyde-3-phosphate dehydrogenase(GAPDH) bound to discrete areas of the membrane. A separate array wasexposed under standard hybridization conditions to each of the three ³²Plabeled cDNA samples and the three arrays were washed under washingconditions specified by the manufacturer. A STORM Phosphoimager system(Molecular Dynamics, Sunnyvale, Calif.) was Used to measure the signalsdue to hybridization of ³²P labeled cDNA to appropriate areas on themembranes. The data are expressed as arbitrary units calculated by thefollowing formula:Arbitrary units=(Cytokine signal−background signal)/(beta-actinsignal−background signal)

Example 2 Molecular Cloning and Expression Pattern of B7-H3-Encoding DNA

The EST databases of NCBI and Human Genome Sciences, Inc. were screenedfor sequences homologous to the DNA sequences encoding the extracellularregions of all published B7 family members. By a PCR-based strategyusing primers based on EST sequences of interest, two open readingframes (orf) encoding two different allelic forms of a B7-like moleculewere identified in a human dendritic cell (DC)-derived cDNA library. Thenucleic acid sequences were confirmed by analyses of independent RT-PCRproducts from human THP-1 and DC library. The two sequences, whichdiffered by a single nucleotide and most likely corresponding to B7-H3alleles, were observed by sequencing several independent clones obtainedfrom several independent PCRs. In one allele (SEQ ID NO:2) (B7H-3.1)nucleotide 496 is a T (FIG. 1) and in the second allele (B7H-3.2) (SEQID NO:4) nucleotide 496 is a C (FIG. 2). At the protein level, B7-H3.1(SEQ ID NO:1) contains a serine residue at position 166 (FIG. 3) andB7-H3.2 (SEQ ID NO: 2) contains a proline residue at position 166 (FIG.4). Except at position 166, B7-H3.1 and B7-H3.2 have identical aminoacid sequences. The B7-H3.1 orf encodes a putative 316 amino acidprotein (B7-H3) that shares identity in its predicted extracellularreceptor-binding domain with human B7-H1 (27%), B7-H2 (25%), B7-1 (21%),and B7-2 (20%) (FIG. 5A). The putative B7-H3 protein contains a typicalsignal peptide in its N-terminus, a single extracellular V-like Igdomain and a single C-like Ig domains, a transmembrane region, and a 45amino acid cytoplasmic tail (FIG. 5B), indicating that B7-H3 is a type Itransmembrane protein belonging to the Ig superfamily. Similar to othermembers of the family, B7-H3 has four conserved cysteine residues thatare believed to be involved in the formation of V- and C-like Igdomains. The absence of a heptad structure and B30.2 domains makes B7-H3distinct from butyrophilins and myelin oligodendrocyte glycoproteins[reviewed in Linsley, et al. (1994) Protein Sci. 3, 1341-1343; Henry etal. (1999) Immunol. Today 20, 285-288]. The above data indicate thatB7-H3 is a member of the B7 costimulatory ligand family.

Northern blot analysis indicated that B7-H3 is encoded by a single4.1-kb mRNA and is expressed at high levels in many normal human tissuesincluding heart, liver, placenta, prostate, testis, uterus, pancreas,small intestine, and colon; low levels of B7-H3 mRNA were also found inbrain, skeletal muscle, kidney, and lung. B7-H3 mRNA could also bedetected in several lymphoid organs including spleen, lymph nodes, bonemarrow, fetal liver, and thymus. Surprisingly, no B7-H3 mRNA wasdetected in RNA from PBMC in two independent northern blots. Severaltumor lines, including melanoma G361, cervix adenocarcinoma HeLa S3,chronic myelogenous leukemia K562, lung carcinoma A546, and colorectaladenocarcinoma SW480, also express B7-H3 mRNA. Molt-4 (lymphoblasticleukemia) and Raji (Burkitts lymphoma) were negative for B7-H3 mRNA.Equal levels of actin mRNA were observed in all the blots. Using RT-PCR,B7-H3 mRNA was detectable in RNA from K562, U937, THP-1, and dendriticcells but not in Raji, Jurkat, Molt-4, or a T cell clone.

FFC analysis was performed to detect cell-surface expression of B7-H3protein. Polyclonal antibodies specific for human B7-H3 were generatedby immunizing mice with KLH-conjugated synthetic peptides spanning thehydrophilic regions of human B7-H3. Results obtained using an antiserumgenerated by immunization with a peptide containing amino acid residues142-165 are described. The antiserum stained 293 cells transientlytransfected to express B7-H3, but not B7-H1, B7-H2, nor B7-1 (FIGS. 6A,7B and data not shown), indicating that this antiserum is specific forB7-H3 protein. B7-H3 is detectable on a small fraction of resting CD14⁺cells (<3%), but not on resting CD3⁺ and CD19⁺ cells (FIG. 6B).Activation (“standard activation”) of PBMC by various methods includingPHA, LPS, or a combination of LPS and IFN-γ did not modulate B7-H3expression. In contrast, stimulation with a combination of PMA andionomycin significantly increased surface expression of B7-H3 on CD3⁺and CD14⁺ cells but not on CD19⁺ cells (FIG. 6B). Interestingly,treatment by PMA and ionomycin (but not by LPS) also increased theexpression of B7-H3 on cytokine-induced DC and on cells of the humanmonocytic tumor line U937 (FIG. 6B). The epithelium-derived tumor linesincluding choriocarcinoma BeWo, colorectal adenocarcinomas HT29, WiDr,and SW620 negative for B7-H3, with or without activation by PMA andionomycin (data not shown). These data indicate that surface B7-H3 isnot constitutively expressed on the majority of hematopoietic cells butis selectively induced by PMA and ionomycin in T cells, monocytes, andDC.

Example 3 Expression of a B7-H3 Counter-Receptor

To determine whether a counter-receptor (B7-H3CR) for B7-H3 is expressedon T cells, a B7-H3Ig fusion protein was prepared by fusing theextracellular domain of B7-H3 and the Fc portion of mouse IgG2a. FFCanalysis (FIG. 7A) indicated that purified resting T cells do notexpress a B7-H3CR. Stimulation of T cells with PHA, however, led to arapid up-regulation of B7-H3CR within the first 24 h of culture. Theexpression waned after 48 h (FIG. 7A).

293 cells were transiently transfected with pcDNA/B7-H3, stained withCTLA4Ig and ICOSIg, and subjected to FACS analysis. The cells were alsostained with anti-B7-H3 antibodies as a positive control. NeitherCTLA4Ig nor ICOSIg stained B7-H3/293 cells (FIG. 7B). Furthermore, whileanti-B7-H3 antibodies did not bind to B7-1/293 or B7-H2/293, CTLA4Igbound to B7-1/293 and ICOSIg bound to B7-H2/293 (FIG. 7B). Thus B7-H3 isa ligand for an inducible T cell counter-receptor distinct from CTLA-4and ICOS.

Example 4 B7-H3 Co-Stimulates T Cell Proliferation and the Generation ofCTL

A previously described co-stimulation assay was used [Dong et al. (1999)Nat. Med. 5, 1365-1369] to test whether B7-H3 co-stimulates theproliferation of T cells. In this assay, purified T cells werestimulated by immobilized anti-CD3 mAb in the presence of immobilizedB7-H3Ig. The proliferation of T cells was determined by[³H]-thymidine-incorporation after a 72 h incubation. B7-H3Ig increasedT cell proliferation in a dose-dependent fashion (FIG. 8A) in thepresence of a suboptimal dose of anti-CD3 mAb (coated onto the plates ata concentration of 40 ng/ml). Interestingly, B7-1Ig coated onto theplates at concentrations of 2.5-10 μg/ml induced significantly higherlevels of T cell proliferation than did B7-H3Ig (FIG. 8A). In theabsence of anti-CD3 mAb, neither B7-H3Ig nor B7-1Ig inducedproliferation of T cells. Immobilized B7-H3Ig significantly enhancedproliferation of both CD4⁺ and CD8⁺ T cells (FIG. 8B).

To evaluate the ability of B7-H3 to affect CTL generation, purifiedhuman T cells from healthy donors were stimulated with cells of themelanoma line 624mel transiently transfected with pcDNA/B7-H3 or controlvector. CTL activity was determined by lysis of ⁵¹Cr-labeled 624melcells. B7-H3/624mel cells induced significantly higher CTL activity thandid mock-transfected 624mel cells (FIG. 8C). Thus B7-H3 co-stimulatesthe growth of both CD4+ and CD8+ T cells and enhances the generation ofCTL.

Example 5 Molecular Cloning and Expression Pattern of B7-H4 Encoding DNA

Using a strategy similar to that described above for B7-H3, the cDNAnucleotide sequence of an orf (SEQ ID NO:6) (FIG. 9) encoding anotherB7-homologue, B7-H4, was identified. This orf encodes a 282 amino acidprotein (B7-H4; SEQ ID NO:5) (FIG. 10) which has significant homology inits predicted extracellular domain with human B7-H1, human B7-H2, humanB7-H3, human B7-1, and human B7-2 (FIG. 11). The B7-H4 protein containsa typical signal peptide in its N-terminus, extracellular V- and C-likeIg domains, a transmembrane region, and a cytoplasmic tail, therebyindicating that B7-H4 is a type I transmembrane protein belonging to theIg superfamily. Similar to the other members of the B7 family, B7-H4 hasfour conserved cysteine residues (marked *) that are believed to beinvolved in the formation of V- and C-like Ig domains. The above datasuggest that B7-H4 is a member of the B7 co-stimulatory ligand family.

Northern blot analysis was performed to determine mRNA expression of theB7-H4 gene. The analysis indicated that B7-H4 mRNA is expressed atrelatively high levels in the lymphoid tissues spleen and thymus andweakly in lung.

Example 6 B7-H4 Co-Stimulates T Cell Proliferation

In order to carry out T cell co-stimulation experiments, fusion proteinscontaining the extracellular domain of B7-H4 and the CH2-CH3 portion ofeither mouse IgG2a heavy chain (B7-H4Ig) or human IgG1 (B7-H4hIg) wereprepared by recombinant methods substantially the same as that describedabove for the production of B7-H3Ig. In the co-stimulation assays, nylonwool purified T cells were stimulated in vitro by immobilized anti-CD3mAb in the presence of the immobilized fusion proteins containing theextracellular domains of the B7-H4 (B7-H4Ig or B7-H4hIg) or B7-1(B7-1Ig). Control culture wells contained immobilized anti-CD3 mAb andcontrol human IgG (hIgG) or contained immobilized anti-CD3 mAb and were“mock” coated with phosphate buffered saline (PBS) (“PBS”). The anti-CD3mAb was coated onto the bottoms of microtiter plate tissue culture wellsat the indicated concentrations (FIG. 12 and FIG. 13) and the fusion orcontrol proteins were coated onto the bottoms of the wells at either 5μg/ml (FIG. 12) or 10 μg/ml (FIG. 13). The assays were carried out asdescribed above for the experiments testing for B7-H3 co-stimulatoryactivity with T cell proliferation being expressed in terms of theamount (“CPM”) of [³H]-thymidine incorporated into the T cells after a72 h culture. While coating at a concentration of 5 μg/ml resulted in asimilar level of T cell co-stimulation with B7-H4Ig, B7-H4hIg, andB7-1Ig (FIG. 12), coating at a concentration of 10 μg/ml resulted insignificantly higher T cell co-stimulation with B7-H4Ig and B7-H4hIgthan with B7-1Ig (FIG. 13). These data indicate that B7-H4 co-stimulatesthe proliferation of T cells.

Example 7 B7-H3 Co-Stimulates the Production Selectively of IFN-γ

Two different assays were used to test whether B7-H3 co-stimulates theproduction of a variety of cytokines. The first assay employed acytokine mRNA array to measure the relative quantities of mRNA encoding23 different cytokines in cells activated with anti-CD3 antibody in thepresence of either control Ig, B7-1Ig, or B7-H3Ig (see above). The dataare shown in FIG. 14. While the presence of B7-H3Ig resulted indetectable increases in mRNA encoding several cytokines (e.g., IL-1b,IL-2, IL-5, IL-6, IL-8, IL-12 (p35), IL-17, and TNF-α), it caused aremarkable increase in the level of IFN-γ mRNA.

A previously described co-stimulation assay was used [Dong et al. (1999)Nat. Med 5, 1365-1369] to test whether B7-H3 co-stimulates theproduction of IFN-γ, IL-2 and IL-10 at the protein level. In this assay,purified T cells were stimulated in vitro by immobilized anti-CD3 mAb inthe presence of immobilized control Ig, B7-1Ig, or B7-H3Ig. After 72 hof culture, supernatants from all the cultures were removed and assayedfor levels of the three cytokines by ELISA. Data obtained with T cellsfrom four healthy human subjects are presented in Table 1; data fromeach individual and means±standard deviation are shown. While thepresence of B7-H3Ig resulted in small but consistent increases in thelevel of IL-10, it resulted in dramatic increases in the level of IFN-γ.

Thus it appears that B7-H3 selectively co-stimulates the production ofIFN-γ by T cells.

TABLE 1 Co-stimulation of cytokine production by B7-1Ig and B7-H3IgCo-stimulatory Amount of Cytokine Produced Polypeptide Subject IL-2(U/ml) IL-10 (ng/ml) IFN-γ (ng/ml) Control Ig 1 0.86 0.14 24.0  2 0.870.13 37.75  3 0.51 0.12 1.31 4 0.39 0.07 7.72 Mean ± 0.66 ± 12   0.12 ±0.02 17.70 ± 8.22  SD B7-1Ig 1 0.81 1.29 23.20  2 0.72 1.63 142.40  31.38 1.03 8.25 4 1.67 0.44 8.50 Mean ± 1.15 ± 0.23 1.10 ± 0.25 0.87 ±0.28 SD B7-H3Ig 1 0.53 0.64 26.61  2 0.52 1.66 247.08  3 0.89 0.8412.58  4 0.84 0.34 10.25  Mean ± 0.69 ± 0.10 0.87 ± 0.28 74.13 ± 57.76SD

Although the invention has been described with reference to thepresently preferred embodiment, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

1. A nucleic acid encoding a fusion protein, the fusion proteincomprising a first domain joined to one or more additional domains,wherein: (a) the first domain consists of an extracellular region andthe nucleic acid segment encoding the extracellular region comprises asequence that hybridizes over its full length, after a wash at 50° C. to65° C. in a buffer containing 0.2×SSC and 0.1% SDS, to the complement ofthe portion of the nucleotide sequence set forth in SEQ ID NO:6 thatencodes the amino acid sequence from cysteine at position 56 of SEQ IDNO:5 to the cysteine at position 130 of SEQ ID NO:5; and (b) the one ormore additional domains each consists of an amino acid sequence that isunrelated to the polypeptide of SEQ ID NO:5.
 2. The nucleic acid ofclaim 1, wherein the nucleic acid segment encoding the extracellularregion hybridizes over its full length, after a wash at 50° C. to 65° C.in a buffer containing 0.2×SSC and 0.1% SDS, to the complement of theportion of the nucleotide sequence set forth in SEQ ID NO:6 that encodesthe amino acid sequence from cysteine at position 56 of SEQ ID NO:5 tothe cysteine at position 225 of SEQ ID NO:5.
 3. An isolated nucleic acidconsisting of: (a) a nucleotide sequence encoding a polypeptideconsisting of an extracellular region comprising an amino acid sequenceextending from the cysteine at position 56 of SEQ ID NO:5 to thecysteine at position 130 of SEQ ID NO: 5; or (b) the complement of thenucleotide sequence of (a).
 4. The nucleic acid of claim 3, wherein theamino acid sequence extends from the cysteine at position 56 of SEQ IDNO:5 to the cysteine at position 225 of SEQ ID NO:5.
 5. A nucleic acidencoding a fusion protein, wherein the fusion protein comprises a firstdomain joined to at least one additional domain, the first domainconsisting of an extracellular region comprising an amino acid sequenceextending from the cysteine at position 56 of SEQ ID NO:5 to thecysteine at position 130 of SEQ ID NO:5.
 6. The nucleic acid of claim 5,wherein the first domain consists of an extracellular region comprisingan amino acid sequence extending from the cysteine at position 56 of SEQID NO:5 to the cysteine at position 225 of SEQ ID NO:5.
 7. The nucleicacid of claim 1, wherein the one or more one additional domainscomprises all or part of an immunoglobulin constant region.
 8. Thenucleic acid of claim 5, wherein the more or more one additional domainscomprises all or part of an immunoglobulin constant region.
 9. A vectorcomprising the nucleic acid of claim
 1. 10. The vector of claim 9,wherein the nucleic acid is operably linked to a regulatory elementwhich allows expression of said nucleic acid sequence in a cell.
 11. Acell comprising the vector of claim
 9. 12. A cell comprising the vectorof claim
 10. 13. A method of producing a fusion protein, the methodcomprising: culturing the cell of claim 12; and purifying the fusionprotein from the culture.
 14. A vector comprising the nucleic acid ofclaim
 5. 15. The vector of claim 14, wherein the nucleic acid isoperably linked to a regulatory element which allows expression of saidnucleic acid sequence in a cell.
 16. A cell comprising the vector ofclaim
 14. 17. A cell comprising the vector of claim
 15. 18. A method ofproducing a fusion protein, the method comprising: culturing the cell ofclaim 17; and purifying the fusion protein from the culture.
 19. Thenucleic acid of claim 1, wherein the nucleic acid segment encoding theextracellular region hybridizes over it full length, after a wash at 50°C. to 65° C. in a buffer containing 0.2×SSC and 0.1% SDS, to thecomplement of the nucleotide sequence set forth in SEQ ID NO:6.
 20. Avector comprising the nucleic acid of claim
 3. 21. A vector comprising anucleic acid consisting of a nucleotide sequence encoding a polypeptideconsisting of an extracellular region comprising an amino acid sequenceextending from the cysteine at position 56 of SEQ ID NO:5 to thecysteine at position 130 of SEQ ID NO: 5, wherein the nucleic acid isoperably linked to a regulatory element which allows expression of saidnucleic acid sequence in a cell.
 22. A cell comprising the vector ofclaim
 20. 23. A cell comprising the vector of claim
 21. 24. A method ofproducing a polypeptide, the method comprising: culturing the cell ofclaim 23; and purifying the polypeptide from the culture.